Autophagy is the process whereby cytoplasmic components including organelles are engulfed by a double membrane structure and targeted for destruction by fusion with a lysosome. This physiologic process occurs as part of normal housekeeping, as a way to recycle proteins, and as a response to injury. It can be used as a mechanism to prevent apoptosis but can also be used to accomplish cell death when apoptosis is blocked. Autophagy plays an important role in the heart's response to ischemia and reperfusion, hypertrophy, and heart failure. We hypothesize that autophagy is selective rather than nonspecific, and that damaged mitochondria may be the preferred target for autophagy in one setting while sarcoplasmic reticulum, contractile elements, or aggresomes may be selective targets in others. We will develop new methods to analyze organelle-selective autophagic flux. Beclin1 is a key regulator of autophagy and contains a Bcl-2-binding domain. We propose to examine the role of Bcl-2 family members in regulating autophagy. We show that Bcl-2 regulation of ER calcium stores also modulates autophagy and we propose to investigate this in greater detail. Mitochondria are targets of autophagy but must undergo fragmentation before they can be engulfed. The regulation of mitochondrial fission and fusion is achieved through a small set of proteins that also modulate apoptosis. We will test the hypothesis that fission proteins modulate autophagy of mitochondria. The existing tools for study of autophagy in vivo are rather limited. Accordingly, we will develop additional in vivo tools, including regulators of autophagy that can be introduced via Tat mediated protein transduction. Autophagy is part of the innate immune response and is upregulated by bacterial lipopolysaccharide (LPS). LPS and the resulting upregulation of TNFalpha have been shown to exacerbate heart failure. We will test the hypothesis that LPS mediated upregulation of autophagy contributes to heart failure. Autophagy is essential for cells to recycle damaged organelles and cytoplasmic components and therefore may play an important protective role after myocardial infarction, but may also contribute to pathologic remodeling. It is important to thoroughly understand autophagy in the heart as it may lead to the development of new therapeutic agents for amelioration of cardiac dysfunction. Public Health Relevance: This project will examine the role of autophagy in the heart using cell lines, primary culture heart cells, and transgenic animal models. Autophagy is the process used by cells to eliminate damaged organelles and protein aggregates. Its role in the heart is poorly understood. Since infection and inflammation increase autophagy and exacerbate heart failure, we hypothesis that autophagy contributes to injury in this context. [unreadable] [unreadable] [unreadable]